The present invention relates to isolated, attenuated viral strains of human parainfluenza virus 2 (HPIV-2), which are useful in live vaccine preparations. These strains exhibit a temperature sensitive and cold adapted phenotype useful for stimulating a protective immune response in an inoculated mammal without producing the severe symptoms caused by the wild type virus.
The human parainfluenza viruses (HPIV), types 1, 2, and 3, are important pathogens in infants and young children. HPIV routinely causes otitis media, pharyngitis, and the common cold. These upper respiratory tract infections (URI) occur commonly and may be associated with lower respiratory infections (LRI) including croup, pneumonia, and bronchiolitis. Primary infection in young children is associated with lower respiratory disease and often leads to hospitalization. As a group, the parainfluenza viruses are the second most common cause of hospital admission for respiratory infection and are second only to respiratory syncytial virus as a significant pathogen in young children. Parainfluenza type 3 is unique among the parainfluenza viruses in its ability to commonly infect young infants less than 6 months of age. Bronchiolitis and pneumonia are common in infants infected with this type; in this regard, HPIV-3 is similar to respiratory syncytial virus. A number of reviews on HPIV have recently been published (Ray and Compans, 1990; Kingsbury, 1991; Henrickson et al., 1994) concerning the various aspects of these virus infections.
HPIV-2 infection occurs in yearly outbreaks in the United States (Downham et al., 1974). This pathogen has a peak incidence in the fall to early winter with a slightly longer “season” than HPIV-1. Croup is the most frequent LRI caused by this virus, but it can also cause any of the other respiratory illnesses associated with HPIV-1. The peak incidence of HPIV-2 infections occurs in the second year of life with approximately 60% of infections taking place in children less than 5 years of age. Of interest is the observation in one study that more girls than boys were symptomatic with LRI caused by HPIV-2, than LRI caused by HPIV-1 or 3 (Downham et al., 1974). LRI caused by HPIV-2 has been reported less frequently than with HPIV-1 and HPIV-3. Recent reports have indicated that either geographic differences or differences in isolation and detection techniques may play a role in under-reporting this virus (Downham et al., 1974; Henrickson et al., 1994). It has been estimated that during the 1991 epidemic, as many as 157,000 children under the age of 5 were seen in emergency rooms, and 35,000 children were admitted to hospitals in the United States with HPIV-2 infection. This epidemic resulted in almost $200 million of direct patient care costs for HPIV-1 and -2 combined.
All of the human parainfluenza viruses are very similar in structural, physicochemical, and biological characteristics. A prototypic HPIV virion is composed of a single RNA strand of negative polarity surrounded by a lipid envelope of host cell origin. These are pleiomorphic, or multi-formed, viruses which have an average diameter of 150 to 250 nm. The typical HPIV genome contains approximately 15,000 nucleotides of genetic information (Storey et al., 1984) and encodes at least six viral proteins (3″-NP-P(+C)M-F-HN-L-5′) (Storey et al., 1984). In addition, HPIV-1, 2, and 3 encode an additional nonstructural protein, “C,” and HPIV-2 a protein “V.” These proteins are produced from overlapping reading frames within the P gene and may require editing of the mRNA (Matsuoka et al., 1991). The complete nucleotide sequence of the HPIV-2 genome has not been published.
The human parainfluenza viruses are classified within the genus Paramyxoviridae. There are five major serotypes within this genus: the HIPV's 1-4, and mumps. The HPIV serotypes can be grouped antigenically into two divisions: (1) HPIV-1 and HPIV-3, within the genus Paramyxovirus, and (2) HPIV-2 and HPIV-4, within the genus Rubulavirus (Collins et al., 1996). HPIVs all share common antigens and variable levels of heterotypic antibody are often detected during infection. Thus, it is difficult to determine whether the heterotypic responses are reflective of past infections, or simply are cross reactions to similar antigens during serologic testing. However, specific hyperimmune animal serum in the past and, more recently, monoclonal antibodies have been employed to differentiate these viruses in standard assays (Sarkkinen et al., 1981).
Mucous membranes of the nose and throat are the initial site of parainfluenza virus infection. Patients with mild disease may have limited involvement of the bronchi as well. The larynx and upper trachea are involved in more extensive infections with HPIV-1 and HPIV-2, and result in the croup syndrome. Infections may also extend to the lower trachea and bronchi, with accumulation of thickened mucus and resultant atelectasis (incomplete lung expansion) and pneumonia. The possible contribution of the immune response to the pathogenesis of this illness is suggested by the observation that infants and children who develop parainfluenza virus croup produce local, virus-specific IgE antibodies earlier and in larger amounts than patients of comparable age who develop infections restricted to the upper respiratory tract (Welliver et al., 1982). Cell-mediated immune responses to parainfluenza viral antigens, as well as parainfluenza virus-specific IgE antibody responses, have been reported to be greater among infants with parainfluenza virus bronchiolitis than among infected infants who develop only upper respiratory illnesses. A prolonged carrier state of HPIV-3 is observed in patients with chronic bronchitis and emphysema (Gross et al., 1973). It has been suggested that healthy adults may shed infectious viruses intermittently and infect susceptible individuals; furthermore, investigators have also suggested that persistent infection might occur (Parkinson et al., 1980).
The hamster provides a recognized animal model for HPIV infection. Infected animals develop recognizable pathologic changes in the lung which are not altered by passive administration of antibodies (Glezen and Femald, 1976). Infected hamsters do not show visible signs of respiratory illness or a significant weight loss during infection. In addition, monkeys may be used as an animal model of infection, as demonstrated in the Examples 4-6, below.
A variety of vaccines have been developed over the years to prevent various viral infections in animals and humans. Two principal types of vaccines have been used: killed viruses and attenuated live virus. A killed virus is typically inactivated by chemical or physical treatment, but is generally less effective in stimulating a lasting immune response than an attenuated live virus. Attenuated live viruses are typically more effective, but may revert back to their virulent state while in the body. The time and cost involved in developing either killed or live vaccines is significant.
Live, attenuated vaccines may be obtained directly from progeny viruses isolated from infected animals. For example, U.S. Pat. No. 3,927,209 discloses a parainfluenza type-3 vaccine isolated as a virus strain from a bovine respiratory tract. Live attenuated vaccines may also be obtained by repeatedly cold passaging a wild-type strain through suitable cultures until the virus has lost its original pathogenic properties. A “cold passage” is the growth of a virus through an entire life cycle (infection of the host cell, proliferation in the host cell, and escape from the host cell) at a temperature lower than that in which the virus normally replicates. For example, cp45, a cold-adapted, temperature sensitive strain was obtained by passing the wild-type virus (JS strain) of HPIV-3 45 times at reduced temperatures. (Belshe and Hissom, 1982). The temperature sensitive cp45 strain is currently under evaluation for use as a candidate vaccine for HPIV-3 in humans. (Karron et al. 1995; Hall et al. 1993; Belshe et al. 1992; Clements et al. 1991; Crookshanks-Newman and Belshe 1986). Recent evaluation in children has revealed the cp45 strain to be highly attenuated and effective in stimulating an immunogenic response. (Karron et al. 1995; Belshe et al. 1992). Although cold passaging techniques have also been used to produce Influenza A and B vaccine strains, no similar successful cold passaging of an HPIV-2 virus has been described.
Attenuation in a particular vaccine strain is commonly evaluated with respect to three phenotypes of the strain: cold adaptation, temperature sensitivity and plaque size or yield in tissue culture. Cold adaptation (ca) relates to the ability of the virus to grow at reduced temperatures around 25° C. and temperature sensitivity (ts) relates to whether such growth is inhibited at temperatures around 40° C. Plaque titers are an assay for quantitatively evaluating the extent of virus growth, and are commonly used to evaluate the extent of cold-adaptive and/or temperature sensitive phenotypes. Other methods for determining whether vaccine is attenuated involve administering the vaccine to primates. For example, the attenuation of new polio vaccine lots is typically tested in monkeys before being approved for sale by the FDA.
Given the propensity of HPIV-2 disease to cause severe respiratory distress in infants and young children, a vaccine which would prevent severe infection, and the resulting necessity for hospitalization and treatment, is very desirable. Although the need for an HPIV-2 vaccine has been recognized for over two decades, and despite successes in isolating vaccine strains for HPIV-3 in the early 1980′s, there is currently no vaccine available to immunize children against HPIV-2. Prior to the discovery of the applicants, HPIV-2 had not been successfully cold passaged. The difficulty in isolating attenuated strains of the HPIV-2 virus, as compared to the HPIV-3 virus, can be explained by the considerable morphological and phenotypic differences between the two viruses. Although they are antigenically similar, HPIV-2 is much more difficult to adapt to in vitro growth conditions and reduced temperatures than HPIV-3.